The potential of kinase-selective phosphoproteomics (phosphokinomics) to provide comprehensive access to the human kinome has previously been demonstrated 
. Our results provide a snap-shot of kinase phosphorylation in NK cells shortly after crosslinking of different activating receptors and identify several kinases not previously implicated in NK cells signaling that are regulated downstream of receptor engagement. To our knowledge, this study is the first to employ phosphokinomics to systematically analyze proximal kinase signaling in primary immune cells.
Phosphokinomics is useful for the systematic characterization of kinase-based signaling on a minute time scale 
. MS-based characterization of phosphorylation events at the level of low abundant protein kinases currently still requires a large amount of cell material and therefore has been restricted to immortalized cell lines. An expansion protocol facilitated the generation of sufficient material (up to 1,5×109
NK cells) for phosphokinome analyses. Importantly, as compared to freshly isolated NK cells, IL-2–cultured NK cells were not significantly altered with respect to the expression of activation receptors, kinetics of intracellular calcium mobilization, or magnitude of degranulation following receptor stimulation. The non-specific kinase inhibitor VI16743 in combination with the commercially available ATP-competitor Purvalanol-B were used to capture the kinome and phosphorylation status of stimulated NK cells. The number of kinases identified by our workflow equaled the number of kinases detected by chemical proteome approaches using a pool of 5 different kinase inhibitors 
and exceeded the amount of kinases captured by VI16743 alone 
. MS data verified the expression of 175 protein kinases (and 13 non-protein kinases) from virtually all branches of the human kinome. Although physiological kinase phosphorylation states and activities in primary cells are probably lower than compared to immortalized cell lines, we obtained quantitative information on more than 300 distinct phosphorylation sites. MS-based analysis of phosphopeptide regulation was assisted by a nano-UPLC system enabling the distinct separation of even isobaric phosphopeptides (identical amino acid sequence but different position of modification). This further supported the unambiguous characterization of phosphorylation sites by high-accuracy MS and the exclusion of compromised iTRAQ reporter signals for their quantitative analyses. Finally, statistical evaluation identified kinases significantly regulated by engagement of CD16 or co-engagement of 2B4 and DNAM-1. Our study revealed 21 kinases on which phosphorylation was modulated upon NK cell receptor stimulation. However, not all of the kinase regulations could be confirmed in each experiment. Besides potential variations in human donors, a limited sensitivity and randomized selection of low-abundant peptide ions during LC-MS experiments always cause some variations in the number of identified phosphopeptides, which then obligatory coincide with missing quantitative information.
Many of the regulated kinases presented in this study have not been previously described in NK cells ( and ). For most regulated sites, phosphorylation induced by CD16 engagement or 2B4 and DNAM-1 co-engagement demonstrated a similar pattern, in agreement with the notion of a convergence of signals for NK cell activation 
. However, PAK4 phosphorylation on S181 was induced by co-stimulation of 2B4 and DNAM-1, but reduced following CD16 engagement (). Preliminary experiments with single receptor stimulations suggest that PAK4 phosphorylation on S181 is induced by 2B4, but not DNAM-1. Thus, PAK4 may be downstream of 2B4 engagement. In T cells, 2B4 can recruit PIX (PAK interacting exchange factor), a PAK-Rac/Cdc42-specific guanine-exchange factor (GEF), via its adapter protein SAP 
. Thus, PAK4 might link proximal 2B4 signaling to actin cytoskeleton rearrangements and be dependent on the SAP/PIX adapter complex. In contrast to PAK4, the phosphokinome approach repeatedly revealed consistent regulation of phosphorylation sites on five kinases (FYN, KCC2, AAK1, FES and MARK2) following stimulation with different receptors.
FYN was consistently phosphorylated upon engagement of all receptors investigated on its N-terminal serines S21, S25 and S26, but phosphorylation of the adjacent tyrosine Y28 was unchanged or even reduced. FYN is known to play an important role in proximal signaling by 2B4 
. Additionally, FYN is involved in DNAM-1-mediated signaling, as has previously been demonstrated in T cells and mast cells 
. A role for FYN in NK cell signaling downstream of CD16 has not been described. FYN phosphorylation on S21 and S25 has been identified by other systematic phosphoproteome approaches, whereas FYN phosphorylation on S26 is novel. Interestingly, recent work suggests that FYN is phosphorylated by PKA on S21 and that this phosphorylation can regulate FYN activity and reduce focal adhesion dynamics 
. Our data support the idea that S21 plays an important functional role: S21 showed a significantly regulated phosphorylation and constituted the most abundant FYN population in comparison to alternative phosphorylations at S25, S26 and Y28. Together, these data point to a central role for FYN in NK cell activation mediated by different activating NK cell receptors.
The calcium/calmodulin-dependent kinase 2 (KCC2, CAMK2) subunits gamma and delta were phosphorylated on S381–S384 and S330 following CD16 engagement or 2B4 and DNAM-1 co-engagement. Furthermore, our data suggests simultaneous phosphorylation of the S287 activation site on KCC2G. Poggi and colleagues have demonstrated that pharmacological inhibition of KCC2 blocks IL-2–cultured NK cell killing of autologous dendritic cells, but not of K562 cells 
. In T cells, TCR engagement induces translocation of KCC2 to the immune synapse where KCC2 facilitates activation of NFκB through phosphorylation of CARMA1 and Bcl10 
. Data also suggest that KCC2 can negatively regulate NFAT activation, diminishing cytokine transcription in T cells 
. Taken together, although our data implicate activation of KCC2 downstream of activating NK cell receptor engagement, it is not clear how KCC2 modulates NK cell effector functions.
Results revealed that AAK1, the ubiquitously expressed AP2 associated kinase 1 implicated in clathrin-mediated endocytosis 
, possesses a high basal serine/threonine phosphorylation in the region T606-S690. Following engagement of CD16 or co-engagement of 2B4 and DNAM-1, phosphorylation of T672-S678 was reduced. Conversely, stimulation induced AAK1 phosphorylation on T620. Thus, this regulation may potentially serve as molecular switch controlling AAK1 function. AAK1 function has not been studied in NK cells. However, following activation, several NK cell receptors have been shown to undergo SRC-dependent internalization 
. Moreover, juxtaposed to an exocytic pathway, endocytosis is a prominent feature of the cytotoxic NK cell synapse 
. The significance and molecular architecture of endocytosis in regard to NK cell cytotoxicity remains to be elucidated.
Stimulation of CD16 or 2B4 and DNAM-1 induced FES phosphorylation of S716 and dephosphorylation of the postulated auto-phosphorylation site Y173 
, suggesting a regulation of FES activity following NK cell receptor engagement. FES is a SRC family kinase involved in cytoskeleton rearrangements 
. In mast cells, FES participates in FcεRI-receptor-induced degranulation 
. Hence, FES might also regulate NK cell degranulation.
MARK2 was consistently dephosphorylated on S456 following engagement of CD16 or co-engagement of 2B4 and DNAM-1. MARK family kinases phosphorylate microtubule-associated proteins, regulate microtubule-based intracellular transport, and are implicated in cellular polarity 
. In T cells, MARK2 has been shown to become phosphorylated on S400 and T595 following TCR engagement and be required for microtubule-organizing center polarization 
. Thus, it will be interesting to investigate how MARK2 might contribute to NK cell cytotoxicity.
This study characterizes a significant portion of the NK cell kinome and provides a first unbiased and systematic view into kinase signaling in primary NK cells. Proteomics detected receptor-dependent kinase phosphorylations already after 2 minutes of receptor engagement and allowed the discrimination of differentially phosphorylated kinase populations as demonstrated by FYN. Several kinases were consistently regulated following the engagement of activating receptors. Co-engagement of DNAM-1 and 2B4 generates a phosphorylation pattern at kinases, which was rather similar to that of CD16-stimulated cells, supporting the idea of a core signal network for NK cell activation. Kinases and phosphorylations, which have not previously been implicated in NK cell signaling were regulated and may thus contribute to the process of NK cell activation. Regulation of kinases such as MARK2 and AAK1 are interesting in terms of understanding the molecular pathways for cellular polarization and endocytosis, processes that are induced upon NK cell activation. Thus, contemplating data presented here will direct future studies focused on elucidating the molecular regulation of NK cell effector functions.